The overall objecitive of this proposal is to investigate the sequential intracellular synthesis and appearance of cholesteryl ester as it is incorporated into intracellular membranes, nascent lipoprotein particles, lipid droplets or other subcellular organelles. Studies will be performed in control and hypercholesterolemic rats. The subcellular localization of acyl CoA:cholesterol acyltransferase will be determined among the intracellular organelles of rat liver normally associated with the microsomal fraction. Emphasis will be given to assaying for acyl CoA:cholesterol acyltransferase in the different subfractions using methodology which considers the influence of varying endogenous cholesterol levels and exogenously added long-chain fatty acyl CoA. Radiolabeled acetate and glycerol will be injected intraperitoneally and the sequential appearance of the newly synthesized cholesteryl ester and triglyceride among the membrane and cisternal contents of the rough endoplasmic reticulum, smooth endoplasmic reticulum. Golgi apparatus and secretory vesicles will be determined. Lipoprotein particles from the cisternal contents will be isolated and characterized. The topological orientation of the cholesteryl ester formed by acyl CoA: cholesterol acyltransferase in the microsomal membrane will be determined using a combination of acid lipase, phospholipase and detergent treatments.